I'm making a transgene that's ~5kb that I'm trying to insert into the Rosa26 locus using the CRISPR/Cas9 system. I blasted a CRISPR guide RNA sequence, that was previously used by Fujii et al, against the mouse genome and although 20 of 20 bases align to the Rosa26 locus, there are several alternative loci that were homologous to the guide RNA at 16-19 bases. Therefore, insertion into these sites is not impossible. I want to propagate single cell clones and evaluate where in the genome the transgene inserted. What's the best way to evaluate this? I was thinking about making a primer at the end of my transgene and performing sanger sequencing downstream; however, if the gene inserted at numerous sites this could look messy. Is there a better method?
Edited by Ahrenhase, 28 February 2014 - 06:28 PM.